We propose to carry out experiments to significantly improve the efficiency of insertional mutagenesis in the mouse, and thereby make it possible to systematically mutate and clone developmentally important genes. By characterizing these genes and defining the molecular actions and interactions of their gene products, we hope to increase our understanding of the basic mechanisms controlling normal mammalian development. Our approach is to use retroviruses to insertionally mutate mouse embryonic stem cells (ES cells). Our specific aim is to develop a strategy for following the inheritance patterns of large numbers of integrated retroviruses at the DNA level. Instead of infecting ES cells with many copies of a single type of vector, we will construct a set of new vectors, each of which has a unique oligonucleotide sequence "label", and introduce multiple copies of each of these several vectors. Although the maximum number of vectors of any one type introduced will be determined by the limits of resolution of the gel system, being able to analyze each type separately will allow the inheritance patterns of a very large total number of proviruses to be followed. Infected ES cells will be used to derive chimeras and mouse strains. Using standard breeding programs, we will screen the transgenic strains for mutants which are in some way disrupted in development and by molecular analysis we will determine the specific proviruses which cosegregate with each new phenotype. We estimate that only 5% of the introduced retroviruses will actually lead to mutations. Because the inheritance patterns of the remaining integrations can also be followed, the entire set can be genetically mapped. It may be possible to establish linkage of certain retroviral integrations with previously identified developmental genes. Depending on the degree of genetic linkage, proviruses not actually causing mutations could still serve as starting points for cloning genes using long range mapping and chromosome jumping methods. Therefore, introducing a large number of different types of retroviral vectors may well provide two distinct routes for cloning developmentally important genes.